Direct intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) is neuroprotective against ischemia-induced cerebral injury. Utilizing viral vectors to deliver and express therapeutic genes presents an opportunity to produce GDNF within localized regions of an evolving infarct. We investigated whether a Herpes simplex virus (HSV) amplicon-based vector encoding GDNF (HSVgdnf) would protect neurons against ischemic injury. In primary neuronal cultures HSVgdnf reduced oxidant-induced injury compared to the control vector HSVlac. To test protective effects in vivo, HSVgdnf or HSVlac was injected into the cerebral cortex prior to or subsequent to a 60 minute unilateral occlusion of the middle cerebral artery (MCA). Control stroke animals developed bradykinesia and motor asymmetry; pretreatment with HSVgdnf significantly reduced such motor deficits. Animals receiving HSVlac or HSVgdnf after the ischemic insult did not exhibit any behavioral improvement. Histological analyses performed one month after stroke revealed a reduction in ischemic tissue loss in rats pretreated with HSVgdnf. Similarly, these animals exhibited less immunostaining for glial fibrillary acidic protein (GFAP) and caspase-3. Taken together, our data indicate that HSVgdnf pretreatment provides protection against cerebral ischemia and supports the utilization of the HSV amplicon for therapeutic delivery of trophic factors to the CNS.